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Tumor Suppressor Genes Gatekeepers and Caretakers “Caretakers” do not directly control cell birth or cell death but rather control the rate of mutations of other genes, including gatekeeper genes. “Gatekeepers” are the genes that directly control cell birth and cell death. RB, p53, PTEN, APC, BRCA1 and BRCA2 XP-A, ATM, hMSH1, hMLH2, hPMS1,hPMS2, WRN-H

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Importance of the cell cycle and apoptosis in tissue homeostasis. TISSUE HOMEOSTASIS The balance between cell birth and cell death! Homeostasis Lost TUMOR Hyperplasia

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Tumor growth kinetics is different for gatekeepers and caretakers When a gatekeeper gene is altered through mutation, the rate of cell birth exceeds that of cell death, and a tumor is initiated. When a caretaker gene is altered, the cell accumulates mutations at a high rate and the process of tumorigenesis is accelerated. A raise in mutation rate may make tumorigenesis faster. The emergence and survival of a tumor is most likely a form “DARWINIAN MICROEVOLUTION” Survival of the fittest!!! A raise in mutation rate may make tumorigenesis faster. The emergence and survival of a tumor is most likely a form “DARWINIAN MICROEVOLUTION” Survival of the fittest!!!

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Retinoblastoma Appearance Retinoblastoma may be unifocal or multifocal. About 60% of patients have unilateral RB with a mean age of diagnosis of 24 months; About 40% have bilateral RB with a mean age of diagnosis of 15 months. Secondary tumors – osteosarcomas and rhabdomyosarcomas 4.4% have secondary tumor in 10 years, 18.3% in 20 years; 26.1 % in 30 years 70% of patients have point mutations in RB1 gene; 10% -- partial deletion of RB1 gene; 20% -- causes unknown; appearance is the same

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Structure of the RB1 gene RB1 gene covers 180-kb in 13q14 region The 27 exons of RB1 range in size from 31 to 1,889 base pairs. The translated product of RB1 (p105-RB1) consists of 928 amino acids. About 80-85% of mutations result in a premature termination codon. Mutations are scattered throughout exon 1 to exon 25 of the RB1 gene and its promoter region. About 80% of de novo germline mutations are paternal in origin. The reason for this is unknown

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Molecular Mechanisms of RB1 How does RB1 control cell cycle progression? RB1 protein sequesters a transcription factor called E2F. The E2F family of transcription factors are required for transcriptional activation of genes needed for DNA metabolism/synthesis. Genes such as thymidine synthatase, dihydrofolate reductase, DNA polymerase alpha, and others

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- a universal inhibitor of CDKs originally - isolated in one of three ways: a p53 inducible transcript (WAF1); interaction with CDK2 (CIP1); mRNA over expressed in senescent cells (SD1) - binds to CDK1, CDK2,3,4 and 6 - normal fibroblasts found in a quaternary complex, throughout the cell cycle, with CDK and PCNA (proliferating cell nuclear antigen) - complex has activity in proliferating cells but is inhibited by the addition of more p21 to the complex p21 WAF1/CIP

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INK4-family of CDK inhibitors p16Ink4a p15Ink4b p18Ink4c p19Ink4d compete with D-type cyclins for binding to the CDK subunit The inhibitory action of the Ink4 proteins is largely dependent on the presence of pRb in the cell. When RB1 is damaged cyclin E expression is already increased and inhibition of cyclin D-CDK4 complexes does not inhibit S-phase entry

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p16 Ink4a Most studied gene in the family because of three reasons: -- Its mutations are common in hereditary and sporadic melanoma samples -- Only this gene out of whole family fulfills all criteria for being tumor suppressor -- Very special genomic structure of this gene